Today many powerful techniques, derived from research in molecular biology, are ready to be used in routine diagnostic or forensic applications. Prominent examples are the polymerase chain reaction, PCR (Saiki, R. K. et al., Science, 230, 1350-1355), based on a cyclic, template-directed primer extension reaction; the analysis of restriction fragment length polymorphisms (Kan, Y. W. and A. M. Dozy, Lancet, 2, 910-912); and the ligase chain reaction (Barany, F. (1991) Proc. Natl. Acad. Sci. USA, 88, 189-193) to detect known point mutations at the ligation site of adjacent oligonucleotides.
It appears likely that in the near future, application of these techniques for analysis of DNA will augment or supplant conventional diagnostic procedures based, for example, on the detection of disease-associated metabolites.
Currently, the most common tool for the analysis of DNA is fragment separation by gel electrophoresis. However, in many cases, electrophoretic analysis and subsequent detection of labeled fragments is more time-consuming than performing the enzymatic reaction, and therefore is a time-limiting step.
Detection techniques are under development which will enhance signal acquisition and provide automated and parallel sample processing, and will likely lead to cost-efficient and time-saving sample processing in diagnostic and forensic applications. Also, large DNA sequencing projects, such as the Human Genome Initiative, that seek to sequence genes or entire genomes, for research or diagnostic purposes, require automated techniques with a high throughput to ensure timely completion of the project.
A promising tool which meets at least some of these criteria is the analysis of DNA fragments by matrix assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (Karas, M. and Hillenkamp, F. (1988) Anal. Chem., 60, 2299-2301).
The biotin-streptavidin system is a common and useful tool for the purification of biotinylated materials (X. Tong and L. M. Smith (1992) Anal. Chem., 64, 2672-2677), e.g., products from PCR or sequencing reactions. Streptavidin (and also avidin) are bacterial proteins which form tight complexes with biotin, including biotin conjugated to other molecules such as nucleic acids. The stability of the biotin-streptavidin complex during intensive washing permits removal of non-specifically bound and non-biotinylated material, which is of great importance for the success of reaction product analysis. The properties of the biotin-streptavidin complex can be used in systems employing biotin bound to streptavidin on a solid support and to yield immobilized biotinylated molecules. The solid phase, including the complexed biotinylated molecules, can be physically collected for further manipulations, including i) removal of excess reaction components like buffer salts, enzymes or deoxynucleotide triphosphates (dNTPs) or ii) performance of enzymatic reactions like nucleolytic digests and solid phase sequencing.
A broad spectrum of applications for the biotin-streptavidin system is known and even techniques not yet developed will be adaptable to this system (see, e.g., Stahl et al. Nucleic Acids Research (1988) 16, 3025-3038; Hultman et al. Nucleic Acids Res. (1989) 17, 4937-4946; Hornes et al. Genet. Anal. (1990) 7, 145-150).
Although the biotin-streptavidin complex is the result of non-covalent bonding, the affinity of streptavidin for biotin is about one million times more powerful than that of most antibody-antigen interactions. However, for the analysis of reaction products it is important to provide conditions for an effective dissociation of the complex while recovering the analyte molecules without modification.
Currently the recovery of biotinylated substances is based on biotin-streptavidin complex dissociation using substances like phenol, urea, or, most preferably 95% formamide at temperatures between 25 and 100.degree. C. (Cocuzza et al., U.S. Pat. No. 5,484,701, 1996).
However, the use of formamide has been shown to be harmful for sample crystallization, a necessary process for MALDI-TOF analysis, and for various enzymatic reactions (e.g. reactions employing alkaline phosphatase). Therefore methods based on the use of formamide are only useful for subsequent gel electrophoretic analysis, but are harmful if enzymatic reactions should be performed involving the isolated material or other analytical tools are to be applied. The endo- or exonucleolytic digest, for example, of PCR products would benefit from a method allowing isolation of single-stranded PCR products which can be digested after purification.
Gel electrophoresis, the time and sample throughput-limiting factor in DNA diagnostics, will be replaced by more efficient techniques in the near future. These applications suggest that efficient methods linking the biotin-streptavidin technology to, for example, MALDI-TOF MS, are strongly required.